Genome sequence of the bialaphos producer Streptomyces sp. DSM 41527 and two putative phosphonate antibiotic producers Streptomyces sp. DSM 41014 and DSM 41981 from the DSMZ strain collection

Abstract Streptomyces sp. DSM 41014, DSM 41527, and DSM 41981 are three strains from the DSMZ strain collection. Here, we present the draft genome sequences of DSM 41014, DSM 41527, and DSM 41981 with a size of 9.09 Mb, 8.45 Mb, and 9.23 Mb, respectively.


ANNOUNCEMENT
Phosphonates are rare and often bioactive natural compounds, many of which are nowadays used commercially as medical therapeutics, herbicides, or pesticides.In the course of screening for new phosphonate antibiotic-producing strains, numerous actinomycete strains from the DSMZ collection were cultivated and their genomes sequenced.Here, we present the annotated genome sequences of three strains, which harbour phosphonate biosynthetic gene clusters (BGCs), including the known phosphinothricin tripeptide (PTT, phosphinothricyl-alanyl-alanine) [1,2] producer strain formerly designated as Streptomyces hygroscopicus SF-1293.PTT, also known as bialaphos, is a potent natural product antibiotic mainly utilised for its herbicidal properties [2].Despite the longstanding reputation of S. hygroscopicus SF-1293 (DSM 41527) as a PTT producer and its deposit in several strain collections, the genome sequence was not publicly available.Furthermore, there was no formal description of this strain in literature and no recognition in the List of Prokaryotic names with Standing in Nomenclature (LPSN) [3].In addition, two Streptomyces strains, DSM 41014 and DSM 41981 were genome sequenced and identified as potential phosphonate-producing strains based on the finding of phosphonate-specific biosynthesis genes in their genomes.
For DNA isolation, DSM 41014, DSM 41527, and DSM 41981 were cultivated on a rotary shaker (180 r.p.m.) for 7 days in ISP two broth medium (DSMZ 65) [4] at 28 °C.Then 500 µl of DNA/RNA Shield buffer (Zymo Research, California, USA) were added to 30-50 mg biomass according to the protocol of MicrobesNG (https://microbesng.com/; Birmingham, B15 2SQ, UK) and shipped to the sequencing facility of MicrobesNG following supplier's instructions.Extraction, purification, quantitative, and qualitative estimation of the DNA, as well as whole genome shotgun sequencing and assembly of sequence reads were performed by the MicrobesNG facility using an Illumina platform with random-PCR library selection and 250 bp paired end protocol.The following methodology is reproduced from the methods supplied by MicrobesNG (accessed on 4 January 2024: https://microbesng.com/documents/39/Genome_Sequencing_Methods_V20231206.pdf).Five to forty microlitres of the cell suspension were lysed with 120 µl of TE buffer, containing lysozyme (MPBio, USA), metapolyzyme (Sigma Aldrich, USA), and RNase A (ITW Reagents, Spain), with subsequent incubation for 25 min at 37 °C.Proteinase K (VWR Chemicals, Ohio, USA) (final concentration 0.1 mg ml −1 ) and SDS (Sigma-Aldrich, Missouri, USA) (final concentration 0.5 % v/v) were added and the mixture was incubated for 5 min at 65 °C.Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (10 mM Tris-HCl, pH 8.0).DNA was then quantified with the Quant-iT dsDNA HS (ThermoFisher Scientific) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom) and diluted as appropriate.Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) according to the manufacturer's protocol with the following modifications: input DNA was increased two-fold, and PCR elongation time was increased to 45 s.DNA quantification and library preparation were carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland).The library was sequenced using an Illumina NovaSeq 6000 (Illumina, San Diego, USA) with 250 bp paired-end reads setting and 30× depth of coverage.Reads were adapter trimmed using Trimmomatic version 0.30 [5] with a sliding window quality cut-off of Q15.The MicrobesNG bioinformatic pipeline included Kraken [6], a system for taxonomic assignment of short DNA sequences and the BWA-MEM software for mapping the reads.De novo assembly was performed on samples using SPAdes version 3.7 [7].Reads were submitted to NCBI's Sequence Read Archive (SRA) and genome assemblies were submitted Fig. 1.Tree inferred with FastME 2.1.6.1 [19] from GBDP distances calculated from genome sequences.The branch lengths are scaled in terms of GBDP distance formula d 5 .The numbers above branches are GBDP pseudo-bootstrap support values >60 % from 100 replications, with an average branch support of 90.7 %.The tree was rooted at the midpoint [20].
to NCBI as Whole Genome Sequencing projects and annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [8].If not indicated otherwise, for all software default settings were applied.All genome-sequence-related data can be found in (Table 1).
The phylogenetic relationship of the strains was analysed with the EzBioCloud database v. 2.1 [9] using the 16S marker gene, which identified DSM 41014 and DSM 41981 as most similar to Streptomyces cupreus PSKA01 T with 99.52 % similarity [10] and DSM 41527 as most similar to Streptomyces lydicus ATCC 25470 T with 99.17 % similarity [11].Phylogenetic relationship based on the full-length genome sequence was carried out with the Type Strain Genome Server (TYGS) v. 1.0 [12,13] (Fig. 1 gives values independent of genome length by dividing the sum of all identities found in high-scoring segment pairs (HSPs) with the overall HSP length [16].A dDDH value below 70 % is indicative for species delineation.
To unravel the genetic potential for secondary metabolite production, the genome sequences of the three strains were analysed with antiSMASH v. 7.0 [17] for the identification of biosynthetic gene clusters (BGCs).A total of 42, 39, and 38 BGCs were identified for DSM 41014, DSM 41527, and DSM 41981, respectively, with the detection strictness set to relaxed.For all three genomes phosphonate BGCs were identified (Fig. 2a, b, c, respectively), which are characterized by the presence of pepM-like genes, known to code for the enzyme phosphoenolpyruvate mutase (PepM), which is the first and essential enzyme of phosphonate biosynthesis [18].Phosphonate BGCs were identified for DSM 41014 (region 14.1) and DSM 41981 (region 1.5) (Fig. 3a, b, respectively).Both phosphonate BGCs contained the same set of annotated genes, indicating they probably encode for the biosynthesis of similar phosphonate compounds.DSM 41527 contained phosphonate biosynthetic genes as part of a larger hybrid BGC (region 17.1) (Fig. 3c), which included 100 % of all known PTT biosynthetic genes also present in Streptomyces viridochromogenes (MIBiG BGC0000406) [2].
Thus, we here present the first draft genome sequence of the bialaphos producer S. hygroscopicus SF-1293 (DSM 41527) and disclose the genetic potential of DSM 41014 and DSM 41981 to synthesize phosphonate natural compounds.